Stool DNA testing for the detection of pancreatic cancer: assessment of methylation marker candidates

BACKGROUND:

Pancreatic cancer (PanC) presents at late stage with high mortality. Effective early detection methods are needed. Aberrantly methylated genes are unexplored as markers for noninvasive detection by stool testing. The authors aimed to select discriminant methylated genes and to assess accuracy of these and mutant KRAS in stool to detect PanC.

METHODS:

Nine target genes were assayed by real‐time methylation‐specific polymerase chain reaction (MSP) in bisulfite‐treated DNA from microdissected frozen specimens of 24 PanC cases and 30 normal colon controls. Archived stools from 58 PanC cases and 65 controls matched on sex, age, and smoking were analyzed. Target genes from fecal supernatants were enriched by hybrid capture, bisulfite‐treated, and assayed by MSP. KRAS mutations were assayed using the QuARTS technique.

RESULTS:

Areas under the receiver operating characteristics curves (AUCs) for tissue BMP3, NDRG4, EYA4, UCHL1, MDFI, Vimentin, CNTNAP2, SFRP2, and TFPI2 were 0.90, 0.79, 0.78, 0.78, 0.77, 0.77, 0.69, 0.67, and 0.66, respectively. The top 4 markers and mutant KRAS were evaluated in stool. BMP3 was the most discriminant methylation marker in stool. At 90% specificity, methylated BMP3 alone detected 51% of PanCs, mutant KRAS detected 50%, and combination detected 67%. AUCs for methylated BMP3, mutant KRAS, and combination in stool were 0.73, 0.75, and 0.85, respectively.

CONCLUSIONS:

This study demonstrates that stool assay of a methylated gene marker can detect PanC. Among candidate methylated markers discriminant in tissue, BMP3 alone performed well in stool. Combining methylated BMP3 and mutant KRAS increased stool detection over either marker alone. Cancer 2011;. © 2011 American Cancer Society.     

Multiple biomarker panels for early detection of ovarian cancer

Ovarian cancer is the eighth most common cause of cancer mortality in women. It is diagnosed in more than 20,000 women in the USA each year and approximately 15,000 women die of the disease annually. The majority of patients are diagnosed with advanced-stage ovarian cancer, as this deadly disease causes minimal and nonspecific symptoms until late in the course of the disease. No standardized screening test exists to reliably detect ovarian cancer. Cancer antigen (CA)-125 is a protein antigen found at abnormally high levels in the blood of many women with ovarian cancer. Most healthy women have CA-125 levels of below 35 units/microl of blood serum. However, a number of noncancerous conditions can cause elevated CA 125 levels, and many women with early-stage ovarian cancer have normal CA-125 levels. Owing to these limitations, this test is not recommended for routine screening in women who are not at high risk or who do not have specific symptoms of the disease. Currently, many researchers are focusing on simultaneous examination of multiple markers to increase sensitivity of the screening test for early detection of ovarian cancer. Analysis of the current literature shows that combining several biomarkers dramatically improves sensitivity of CA-125 in ovarian cancer patients. This article provides a comprehensive overview of existing studies in the area of multimarker panel development for the early detection and monitoring of ovarian cancer. Our literature review demonstrates that a multimarker approach for the generation of a prototype assay for early detection of ovarian cancer has a great potential to lead to the development of a screening test for this disease.     

Biomarkers for early detection of colon cancer.

There is an increasing demand for biomarkers in colon cancer for risk assessment, early detection, prognosis, and surrogate end points. A number of biomarkers have been identified for early detection of colon cancer, although the risk factors have not been identified extensively. The major advances in understanding colorectal cancer include the identification and the involvement of APC, p53, and Ki-ras in the development and progression of the disease, the identification of the aberrant crypt foci as an early preinvasive lesion, and its relation to the development of cancer. Detecting malignant neoplasms in the early stages offers clinical advantages; therefore, the National Cancer Institute has established an Early Detection Research NETWORK: The emphasis of the network is on translational research and collaboration among scientists.     

Tumor-specific methylation in saliva: a promising biomarker for early detection of head and neck cancer recurrence.

PURPOSE: Our goal was to define tumor and saliva gene methylation profile of head and neck squamous cell carcinoma and to evaluate its prognostic significance and its biomarker potential for early detection of relapse. EXPERIMENTAL DESIGN: We prospectively analyzed 11 genes by methylation-specific PCR on primary tumors, histologically normal adjacent mucosa, and saliva from 90 French patients at diagnosis and during follow-up as well as on 30 saliva specimens from control-matched patients with nonmalignant head and neck pathology. Five additional genes were analyzed on 50 tumors of the series. RESULTS: Methylation of TIMP3, ECAD, p16, MGMT, DAPK, and RASSF1 was the most frequently observed in tumors and paired saliva samples were analyzed at diagnosis, with an excellent agreement between both samples. At least one of these six genes was methylated in >75% of the samples without additional positive samples when other genes were analyzed. Methylation profile was similar in newly diagnosed and second primary cancers. Aberrant methylation was not associated with a worse prognosis. Ninety percent of normal adjacent mucosa and all control saliva samples were negative. Twenty-two patients were followed after treatment; abnormal methylation was detectable in the saliva of five patients few months before clinical and 2-deoxy-2[(18)F]fluoro-d-glucose-positron emission tomography signs of relapse, allowing curable surgery. Saliva samples were negative for the 17 other patients: 16 were in remission and only 1 relapsed. CONCLUSIONS: Gene methylation in saliva is a promising biomarker for the follow-up and early detection of still curable relapses of head and neck squamous cell carcinoma patients.     

BAGE hypomethylation, a new epigenetic biomarker for colon cancer detection.

Early detection of colorectal cancer is a decisive step in the successful and complete cure of the disease. Epigenetic markers, in particular, those based on aberrant DNA methylation, can be used to diagnose cancer. B melanoma antigens (BAGE) are a family of genes and truncated genes located in the heterochromatic regions of several human chromosomes. Our previous work showed that BAGE loci (i.e., genes and truncated genes) were hypermethylated in normal tissues and hypomethylated in 98% of human cancers. In the present study, we analyzed DNA methylation of the BAGE loci in 54 colon cancers and in neighboring histopathologic normal tissue samples. Using a combined bisulfite restriction assay, we showed that BAGE loci were hypomethylated in 81% of carcinoma samples. Colon cancer could be diagnosed with 94% specificity, 83% sensitivity, and 89% accuracy. No correlation was found between DNA methylation of BAGE loci and age, gender of patients, nor with the tumor stage or site. Based on the hypothesis that during neoplastic transformation, hypomethylation occurs in juxtacentromeric CpG islands, we suggest that other genes located in the heterochromatic compartment should be tested. These new markers enrich the list of currently studied epigenetic alterations in colon cancer and could be associated with hypermethylation markers to develop reliable diagnostic tests.     

Identification of the methylation of p14ARF promoter as a novel non-invasive biomarker for early detection of lung cancer

Background

Recent diagnostic procedure advances have greatly improved early lung cancer detection. However, the invasive, unpleasant and inconvenient nature of current diagnostic procedures limits their application. There is a great need of novel non-invasive biomarkers for early lung cancer diagnosis. In the present study, we intend to determine whether the blood signatures of p14ARF promoter methylation are suitable for early detection of lung cancer.

Methods

The study aimed to assess the probability of p14ARF promoter methylation in plasma samples to detect early lung cancer using nested methylation-specific PCR in the training set consisted of tumor tissues and paired blood. Besides, we were further to discuss the difference in time to progression between methylation and unmethylation of p14ARF promoter using univariate and multivariate analysis.

Results

The methylation of p14ARF promoter was detected in 33.6 % of tumor tissues, and 12.1 and 25.2 % in distant-cancer mucosa and matched plasma, respectively, and our study has also demonstrated the positive correlation between them by Pearson’s test (r = 0.300). The tumor-free survival time of the unmethylation of p14ARF promoter is significantly longer than that of the methylation of p14ARF promoter in tumor tissues (χ ² = 7.149, P = 0.008).

Conclusion

The methylation of p14ARF promoter in plasma samples has strong potential as a novel non-invasive biomarker for early detection of lung cancer, and the methylation of p14ARF promoter was considered as prognostic factor in our study.     

Reprimo as a potential biomarker for early detection in gastric cancer

PURPOSE: Gastric cancer is a curable disease if diagnosed at early stage. However, most cases are diagnosed at advanced stage because of the lack of screening programs. Therefore, the identification of plasma biomarkers for early detection is necessary. EXPERIMENTAL DESIGN: To search for these biomarkers, we evaluated the DNA methylation patterns of 24 genes by Methylation-specific PCR in primary tissues from 32 retrospectively collected gastric cancer cases (testing group). Correlation between methylation and gene expression was evaluated in the MKN-45 cell line after treatment with 5-aza-2'-deoxycytidine. The most frequently hypermethylated genes were next evaluated in primary tissues and plasma samples from 43 prospectively collected gastric cancer cases as well as plasma samples from 31 asymptomatic age- and gender-matched controls (validation group). RESULTS: In the testing group, 11 genes were hypermethylated in at least 50% of cases (APC, SHP1, E-cadherin, ER, Reprimo, SEMA3B, 3OST2, p14, p15, DAPK, and p16). Eight genes (BRCA1, p73, RARbeta, hMLH1, RIZI, RUNX3, MGMT, and TIMP3) were statistically associated with a particular variant of gastric cancer, the signet-ring cell type (P = 0.03). Seven genes (APC, SHP1, E-cadherin, ER, Reprimo, SEMA3B, and 3OST2) were next evaluated in the validation group. We confirm the high frequency of methylation in primary tumors for all seven genes. However, only APC and Reprimo were frequently methylated in pair plasma samples. In asymptomatic controls, only Reprimo was infrequently methylated in comparison with plasma from gastric cancer cases (P < 0.001). CONCLUSION: Our results identified specific methylation profile associated to signet-ring cell-type histology and aberrant hypermethylation of Reprimo as a potential biomarker for early detection of gastric cancer.     

血浆 MAL 基因甲基化检测对于肺癌早期诊断的意义

目的：研究肺癌患者、肺结核患者及健康人群 T 淋巴细胞相关蛋白（T －lymphocyte maturation －asso-ciated protein,MAL）基因启动子区的甲基化状态,进一步探讨血浆 MAL 基因甲基化检测对于肺癌早期诊断及鉴别肺癌与肺结核疾病的意义。方法：采用甲基化特异性 PCR（methylation special PCR,MSP）方法,检测75例肺癌组织标本、癌旁组织标本和对应的血浆标本及58例肺结核组织标本及对应的血浆标本的 MAL 基因启动子区甲基化状态;同时检测30例正常人群的血浆标本 MAL 甲基化状态作为对照。结果：肺癌组织和癌旁组织中 MAL 基因甲基化发生率分别为78．7％（59／75）和2．7％（2／75）,差别具有统计学意义（P ＜0．001）;肺癌和肺结核组织中 MAL 基因甲基化发生率分别为78．7％（59／75）和3．4％（2／58）,差别具有统计学意义（P＜0．001）;肺癌患者与肺结核患者对应的血浆中 MAL 基因甲基化发生率分别73．3％（55／75）和1．7％（1／58）,差别也具有统计学意义（P ＜0．001）。正常健康人群的血浆标本中未发现 MAL 基因甲基化改变;肺癌组和肺结核组分别与健康人群组比较,肺癌组明显高于健康人群（P ＜0．001）,而肺结核组患者与健康人群比较无明显差异（P ＝0．47）。组织标本和血浆标本的检出率具有一致性（P ＞0．05）。根据病理类型、TNM分期、性别、吸烟史等对肺癌患者进行分组,各组之间均无明显差异（P ＞0．05）。结论：MSP 法检测血浆 MAL 基因甲基化状态,可以作为一种有潜力的肺癌早期诊断方法,对于鉴别肺癌和肺结核也有一定的帮助。     

Modulation of DNA hypomethylation as a surrogate endpoint biomarker for chemoprevention of colon cancer

Surrogate end-point biomarkers are being developed as indicators of the efficacy of chemopreventive agents. These biomarkers are molecular and biological end-points that can be modulated by chemopreventive agents in accordance with their efficacy to prevent cancer. DNA hypomethylation is a common alteration found in colon tumors that has the potential of being modulated by chemopreventive agents and thus being useful as a surrogate end-point biomarker. Agents that were either effective or ineffective in preventing colon cancer were evaluated for the ability to modulate DNA hypomethylation in azoxymethane-induced colon tumors in male F344 rats. DNA methylation was determined by Dot Blot Analysis using a mouse monoclonal anti-5-methylcytosine antibody. Colon tumors had a 70% reduction in DNA methylation relative to normal colonic mucosa. DNA methylation in the tumors was increased by 7 days of treatment with agents that have been shown to prevent colon cancer (calcium chloride, alpha-diflouromethylornithine [DFMO], piroxicam, and sulindac), whereas agents shown not to prevent colon cancer in rats (low dose aspirin, 2-carboxyphenyl retinamide [2-CPR], quercetin, 9-cis retinoic acid, and rutin) did not increase DNA methylation. The results suggest that the ability to reverse the DNA hypomethylation in colon tumors could be useful as a surrogate end-point biomarker for chemoprevention of colon cancer.     

Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer.

PURPOSE: The purpose of this study is to evaluate the usefulness of serum DNA methylation of five tumor suppressor genes for early detection of lung cancer. EXPERIMENTAL DESIGN: Methylation status in serum DNA from 200 patients undergoing bronchofiberscopic examination for abnormal findings on chest radiograph detected by lung cancer screening or surveillance was examined using methylation-specific PCR. RESULTS: Ninety-one patients were given a pathologic diagnosis of lung cancer, 9 other malignant diseases, and 100 nonmalignant pulmonary diseases. In patients with lung cancer, methylation was detected in 18.7% for MGMT, 15.4% for p16(INK4a), 12.1% for RASSF1A, 11.0% for DAPK, and 6.6% for RAR-beta, which was higher compared with that in patients with nonmalignant diseases. Age and smoking status seemed to associate with methylation status. Sensitivity, specificity, and predictive value of methylation in at least one gene for diagnosis of lung cancer were 49.5%, 85.0%, and 75.0%, respectively. Adjusted odds ratio (95% confidence interval) for having lung cancer was 5.28 (2.39-11.7) for patients with methylation in one gene and 5.89 (1.53-22.7) for those with methylation in two or more genes. It is of note that methylation was identified in 50.9% of stage I lung cancer patients, whereas serum protein tumor markers were positive in 11.3% of them. CONCLUSIONS: These results suggest that identification of promoter methylation of tumor suppressor genes in serum DNA could be useful for early detection of lung cancer.     

Improving early breast cancer detection: focus on methylation

The need for additional breast cancer screening tools is indisputably high, as one may conclude from the high rate of interval malignancies in women undergoing regular screening. DNA promoter methylation frequently occurs during breast carcinogenesis and is an early event in this process. Moreover, a field defect for methylation has been described and methylation values can reliably be assessed in limited amounts of DNA. Simultaneous detection of methylation of a panel of genes in breast fluids and/or blood derivatives could be both sufficiently specific and sensitive to be of additive value to current imaging-based screening methods. This review describes the recent developments in methylation detection in breast fluids, serum and plasma that paved the way for large prospective studies. These studies will provide us with the definite answer as to what will be the additive value of defining the methylation status of specific genes to current imaging-based screening methods.     

Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model

Mouse models of intestinal tumors have advanced our understanding of the role of gene mutations in colorectal malignancy. However, the utility of these systems for studying the role of epigenetic alterations in intestinal neoplasms remains to be defined. Consequently, we assessed the role of aberrant DNA methylation in the azoxymethane (AOM) rodent model of colon cancer. AOM induced tumors display global DNA hypomethylation, which is similar to human colorectal cancer. We next assessed the methylation status of a panel of candidate genes previously shown to be aberrantly methylated in human cancer or in mouse models of malignant neoplasms. This analysis revealed different patterns of DNA methylation that were gene specific. Zik1 and Gja9 demonstrated cancer‐specific aberrant DNA methylation, whereas, Cdkn2a/p16, Igfbp3, Mgmt, Id4, and Cxcr4 were methylated in both the AOM tumors and normal colon mucosa. No aberrant methylation of Dapk1 or Mlt1 was detected in the neoplasms, but normal colon mucosa samples displayed methylation of these genes. Finally, p19^Arf, Tslc1, Hltf, and Mlh1 were unmethylated in both the AOM tumors and normal colon mucosa. Thus, aberrant DNA methylation does occur in AOM tumors, although the frequency of aberrantly methylated genes appears to be less common than in human colorectal cancer. Additional studies are necessary to further characterize the patterns of aberrantly methylated genes in AOM tumors. © 2009 Wiley‐Liss, Inc.     

Mitochondrial DNA mutations in preneoplastic lesions of the gastrointestinal tract: A biomarker for the early detection of cancer

Background  

Somatic mutations of mitochondrial DNA (mtDNA) are common in many human cancers. We have described an oligonucleotide microarray ("MitoChip") for rapid sequencing of the entire mitochondrial genome (Zhou et al, J Mol Diagn 2006), facilitating the analysis of mtDNA mutations in preneoplastic lesions. We examined 14 precancerous lesions, including seven Barrett esophagus biopsies, with or without associated dysplasia; four colorectal adenomas; and three inflammatory colitis-associated dysplasia specimens. In all cases, matched normal tissues from the corresponding site were obtained as germline control. MitoChip analysis was performed on DNA obtained from cryostat-embedded specimens.     

DNA methylation predicts recurrence from resected stage III proximal colon cancer

BACKGROUND:

In colorectal cancer (CRC), DNA methylation anomalies define distinct subgroups termed CpG island methylator phenotype 1 (CIMP1), CIMP2, and CIMP‐negative. The role of this classification in predicting recurrence and disease‐free survival (DFS) in resected stage III CRC was evaluated.

METHODS:

Sporadic cancers from 161 patients were analyzed. Bisulfite pyrosequencing was used to examine the methylation of 2 global DNA methylation markers (LINE‐1, Alu) and 9 loci (MINT1, MINT2, MINT31, P16, hMLH1, P14, SFRP1, SFRP2, and WNT5A). Mutations in BRAF and KRAS were assayed.

RESULTS:

Gene hypermethylation clustered in discrete groups of patients, indicating the presence of CIMP. K‐means clustering analysis identified 3 discrete subgroups: CIMP1 (n = 22, 13.7%), associated with proximal location and BRAF mutations; CIMP2 (n = 40, 24.8%), associated with KRAS mutations; and CIMP‐negative (n = 99, 61.5%), associated with distal location. In proximal CRC, CIMP1 was correlated with a higher recurrence rate (53% for CIMP1, 18% for CIMP2, and 26% for CIMP‐negative) and a worse DFS (P = .015). Also in proximal CRC, LINE‐1 methylation was lower in patients whose cancer recurred compared with those whose cancer did not recur (P = .049). In multivariate analysis, CIMP1 and low LINE1 methylation were independent prognostic factors for DFS in proximal CRC (P = .008 for classification by K‐means clustering analysis; P = .040 for LINE‐1 methylation status).

CONCLUSIONS:

DNA methylation is a useful biomarker of recurrence in resected stage III proximal but not distal CRC. However, as the number of CIMP1 cases was small in distal CRC, further study is required to validate our findings. Cancer 2011. © 2010 American Cancer Society.     

Protein Z: A putative novel biomarker for early detection of ovarian cancer

Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded‐evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down‐regulated up to 2 years pre‐diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up‐regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA‐125 and CA‐125 combined with Protein Z showed a statistically significant (p= 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA‐125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes.     

Targeted methylation sequencing of plasma cell-free DNA for cancer detection and classification

BackgroundTargeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis.Patients and methodsWe developed a comprehensive methylation sequencing assay targeting 9223 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we carried out a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small-cell lung cancer (NSCLC), breast cancer or melanoma and compared results with patients’ outcomes.ResultsMedian methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 versus 85.29; P = 0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 57 (83.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (78.9%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 versus 4.4 months, P < 0.001) and longer time-to-treatment failure (2.8 versus 1.6 months, P = 0.016).ConclusionsComprehensive targeted methylation sequencing of 9223 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.